Project description:We describe the genome-wide DNA-binding of GATA6 in a human CRC cell line (LS174T). GATA6 is found to bind the promoter of genes involved in the maintenance of intestinal stem cells, including genes of the Wnt and TGFbeta/BMP pathways. With this we describe a novel GATA6-dependent mechanism of stem cell maintenance in colorectal tumors.
Project description:We describe the genome-wide DNA-binding of GATA6 in a human CRC cell line (LS174T). GATA6 is found to bind the promoter of genes involved in the maintenance of intestinal stem cells, including genes of the Wnt and TGFbeta/BMP pathways. With this we describe a novel GATA6-dependent mechanism of stem cell maintenance in colorectal tumors. Examination of GATA6 binding and H3K4me1, H3K4me3 and H3K27ac levels in a human CRC cell line by Chromatin immunoprecipitation followed by deep sequencing.
Project description:Deregulated expression of MYC is a driver of colorectal carcinogenesis, necessitating novel strategies to inhibit MYC function. The ubiquitin ligase HUWE1 (HECTH9, ARFBP1, MULE) associates with both MYC and the MYC-associated protein MIZ1. We show here that HUWE1 is required for growth of colorectal cancer cells in culture and in orthotopic xenograft models. Using high throughput screening, we identify small molecule inhibitors of HUWE1, which inhibit MYC-dependent transactivation in colorectal cancer cells, but not in stem and normal colon epithelial cells. Inhibition of HUWE1 stabilizes MIZ1. MIZ1 globally accumulates on MYC target genes and contributes to repression of MYC-activated target genes upon HUWE1 inhibition. Our data show that transcriptional activation by MYC in colon cancer cells requires the continuous degradation of MIZ1 and identify a novel principle that allows for inhibition of MYC function in tumor cells. MIZ1 and MYC ChIPseq experiments in HUWE1 inhibitor-treated Ls174T cells as well as RNAseq experiments in HUWE1- or MIZ1-depleted Ls174T cells after HUWE1 inhibitor treatment. Sequencing was performed on an Illumina Genome Analyzer IIx.
Project description:To validate the suitability of two commonly used colorectal cancer cell lines, DLD1 and SW480, as model systems to study colorectal carcinogenesis, we treated these cell lines with beta-catenin siRNA and identified beta-catenin target genes using DNA microarrays. The list of identified target genes was compared to previously published beta-catenin target genes found in the PubMed and the GEO databases. Based on the large number of beta-catenin target genes found to be similarly regulated in DLD1, SW480 and LS174T as well as the large overlap with confirmed β-catenin target genes, we conclude that DLD1 and SW480 colon carcinoma cell lines are suitable model systems to study beta-catenin regulated genes and signaling pathways 12 arrays (2 cell lines, 2 treatments, 3 biological replicates)
Project description:Aberrant activation of WNT signaling and loss of BMP signals represent the two main alterations leading to the initiation of colorectal cancer (CRC). Here we screen for genes required for maintaining the tumor stem cell phenotype and identify the zinc-finger transcription factor GATA6 as key regulator of the WNT and BMP pathways in CRC. GATA6 directly drives the expression of LGR5 in adenoma stem cells while it restricts BMP signaling to differentiated tumor cells. Genetic deletion of Gata6 in mouse colon adenomas increases the levels of BMP factors, which signal to block self-renewal of tumor stem cells. In human tumors, GATA6 competes with beta-catenin/TCF4 for binding to a distal regulatory region of the BMP4 locus that has been previously linked to increased susceptibility to develop CRC. Hence, GATA6 creates a permissive environment for tumor stem cell expansion by controlling the major signaling pathways that influence CRC initiation. Total RNA was collected from biological replicates of LS174T colorectal cancer cells transduced with either short hairpin against GATA6 (shGATA6) or non-silencing control (NS) grown in DMEM supplemented with 10% FBS in the presence of doxycycline (dox: 1 µg mL-1; Sigma Aldrich) or vehicle (70% ethanol) for a period of 72h. Short hairpins were designed and cloned into doxycycline inducible pTRIPZ lentiviral vector (Open Biosystems). Additionally a 4-hydoxy tamoxifen inducible system was used to inducibly block beta-catenin/TCF activity for a period of 36h. Briefly, the ERT2 domain from the pCMV-CRE-ERT2 (Feil et al., 1996) was amplified by PCR and cloned into a modified FUGW lentiviral vector backbone (Lois et al., 2002). NTCF was then amplified byPCR from the pCDNA3.1-NTCF-NLS (van de Wetering et al., 2002) and cloned in frame upstream of the ERT2 from the FUW-CMV-ERT2 (ET) to create the FUW-CMV-NTCF-ERT2 (NET). Total RNA was extracted using the TRIzol® Plus RNA Purification Kit (Life Technologies).
Project description:GATA6 is a zinc finger transcription factor that is required for the proliferation, development and specific gene regulation in the gastrointestinal tract. We have recently reported that GATA6-mediated induction of the intestinal stem cell marker LGR5 is required for the tumorigenicity of colon cancer cells. However, knockdown of LGR5, unlike GATA6, does not affect the proliferation of these cells under adherent conditions. Here we show that REG4, a member of the regenerating islet-derived (REG) family, is a target of GATA6. We further demonstrate that REG4 is downregulated by overexpression of miR-363, which suppresses GATA6 expression. Moreover, we show that GATA6-mediated activation of REG4 causes an acceleration of the growth of colon cancer cells under adherent conditions. These results suggest that GATA6 simultaneously activates the transcription of genes required for growth (REG4) and clonogenicity (LGR5), and the miR-363-GATA6-REG4/LGR5 pathway is critical for colorectal tumorigenesis. HT29 cells were transfected with a siRNA targeting GATA6.
Project description:GATA6 is a zinc finger transcription factor that is required for the proliferation, development and specific gene regulation in the gastrointestinal tract. We have recently reported that GATA6-mediated induction of the intestinal stem cell marker LGR5 is required for the tumorigenicity of colon cancer cells. However, knockdown of LGR5, unlike GATA6, does not affect the proliferation of these cells under adherent conditions. Here we show that REG4, a member of the regenerating islet-derived (REG) family, is a target of GATA6. We further demonstrate that REG4 is downregulated by overexpression of miR-363, which suppresses GATA6 expression. Moreover, we show that GATA6-mediated activation of REG4 causes an acceleration of the growth of colon cancer cells under adherent conditions. These results suggest that GATA6 simultaneously activates the transcription of genes required for growth (REG4) and clonogenicity (LGR5), and the miR-363-GATA6-REG4/LGR5 pathway is critical for colorectal tumorigenesis. HT29 cells were transfected with a siRNA targeting LGR5.